Joint Application of NMR and Scattering Haydyn Mertens PhD Methods - - PowerPoint PPT Presentation

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Joint Application of NMR and Scattering Haydyn Mertens PhD Methods - - PowerPoint PPT Presentation

Dec 10, 2023 214 likes •662 views

Joint Application of NMR and Scattering Haydyn Mertens PhD Methods combining SAXS with NMR Filtering/scoring NMR conformers Direct refinement of structure Refinement of domain/subunit positions Restraints: SAXS & NMR Orientation

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Joint Application of NMR and Scattering

Haydyn Mertens PhD

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Filtering/scoring NMR conformers Direct refinement of structure Refinement of domain/subunit positions

Methods combining SAXS with NMR

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Restraints: SAXS & NMR

Shapes/envelopes Dimensions (Rg, Dmax) Orientation Interface RDC s PCS NOEs Chemical Shifts PRE

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Restraints: SAXS & NMR

Distances: NOEs Chemical Shifts PRE Orientation: RDCs PCS SAXS provides shape/distance info

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Model Filtering

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Model filtering by SAXS

Structures determined X-ray crystallography NMR Homology modeling Docking algorithms Score these models using SAXS data best fit to SAXS curves cluster models

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Model filtering by SAXS

pyDockSAXS FTDock pyDock SAXS (CRYSOL)

~ 40% improvement in selection of correct solution

Pons et al., J Mol Biol. 2010 Oct 22;403(2):217-30

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Docking Steps: pyDockSAXS

FTDock (http://www.sbg.bio.ic.ac.uk/docking/ftdock.html) generates large pool of complexes Surface complementarity Electrostatics filter pyDOCK (http://www.cllgenome.es/servlet/pydock/) scores/ranks FTDock complexes Binding energy electrostatics desolvation vdw CRYSOL (http://www.embl-hamburg.de/biosaxs/atsas-online/) scores/ranks complexes (fit to SAXS)

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Docking Performance: pyDockSAXS

Identification of near native solutions Combined pyDOCK + CRYSOL is best!

Pons et al., J Mol Biol. 2010 Oct 22;403(2):217-30

43% 29% 21% 23%

Rank (top N predictions for each benchmark member)

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Docking Performance: pyDockSAXS

Anisometry has an impact on selection Spherical complexes hard to select by SAXS

Pons et al., J Mol Biol. 2010 Oct 22;403(2):217-30

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Model filtering by SAXS

pyDockSAXS combined scoring function: pyDockSAXS = EpyDOCK + wcXCRYSOL

Pons et al., J Mol Biol. 2010 Oct 22;403(2):217-30

SAXS can aid in the selection of near-native docking models

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Building a model with RDCs & SAXS

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Building a model with RDCs & SAXS Basic idea: Use known domain structures Orient domains with RDCs Reduce possible solutions with SAXS domain-distance (Rg) scoring/filtering

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Example: Calmodulin

Calmodulin (CaM) Ca2+ binding protein (4 EF-hand motifs) Binds multiple targets (multiple functions) Structure is flexible Solved in both extended & compact forms

Extended form Compact form +ligand

  • ligand
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Example: Calmodulin-TFP (trifluoperazine)

Mattinen et al., Biophys J. (2002) 83:1177-1183

CaM compacts upon TFP binding decreased Dmax observed by SAXS Quaternary structure built from RDCs, domain structures and SAXS

+TFP

SAXS: Rg = 2.0 nm -> Rg = 1.8 nm

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Building CaM-TFP model

Mattinen et al., Biophys J. (2002) 83:1177-1183

RDCs determine PAS (Azz, Axx, Ayy) DNH from single alignment medium Orientations using free CaM domain structures

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Building CaM-TFP model

Mattinen et al., Biophys J. (2002) 83:1177-1183

4 degenerate relative orientations SAXS constrains distance between domains (Rg) Reduced to a single solution

SAXS 1LIN Solutions scored using CRYSOL

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SASREF can do this too!

(in principle)

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Using RDCs in SASREF

Collect RDCs Define domain orientations PALES (http://www.mpibpc.mpg.de/groups/zweckstetter/_links/software_pales.htm) Xplor-NIH python tools (http://nmr.cit.nih.gov/xplor-nih/) Use pre-oriented PDBs as input rigid bodies Allow only translations in SASREF

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Direct Refinement

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Requires accurate and fast calculation of scattering intensity from structure What people currently use: Spherical harmonics (CRYSOL) Zernike potentials (SASTBX) Debye (Xplor-NIH, MODELLER)

Direct Refinement

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Debye:

Quick reminder: intensity calc

Form factors Distances

Need to evaluate all distances Computationally expensive

scales quadratically with number of atoms

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Improve speed: globbic approximation group atoms (eg. GASBOR)

Quick reminder: intensity calc

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Multipole expansion (spherical harmonics) Very fast Computation time is linear with size

Quick reminder: intensity calc

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Direct refinement with NMR & SAXS slow back-calculation of I(s) globbic approx can help reduce number of points bin experimental data less points for calc I(s) Excluded volume treatment Hydration layer treatment

Quick reminder: intensity calc

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Xplor-NIH

(direct refinement)

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  • E. coli Enzyme-I : HPr complex

Active sugar-phosphate transfer in bacteria Phosporylation of E1 and transfer to HPr Significant differences in crystal and NMR structures

Xplor-NIH example:

Schwieters et al., JACS (2010) 132:13026-13045

E1 crystallographic dimers NMR Crystal

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Strategy: backbone n-h RDCs show E1nter subdomain

  • rientations unchanged (free vs complex)

thus assuming E1cter dimerisation domain structure unchanged from E1 crystal structures:

  • rientation E1 dimer from E1nter RDCs

Combine with SAXS/WAXS data Determine free E1 Introduce HPr and determine E1:HPr

Xplor-NIH example:

Schwieters et al., JACS (2010) 132:13026-13045

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Backbone n-h RDCs show E1nter subdomain

  • rientations unchanged (free vs complex)

Xplor-NIH example:

Schwieters et al., JACS (2010) 132:13026-13045

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Xplor-NIH: protocol

E1 dimer E1-HPr dimer

Schwieters et al., JACS (2010) 132:13026-13045

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Comparison to existing structures:

Schwieters et al., JACS (2010) 132:13026-13045

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Xplor-NIH example:

Schwieters et al., JACS (2010) 132:13026-13045

Using SANS to discriminate between 2 possible E1-HPr clusters?

2H-E1/1H-HPr in 40% D2O (see E1 only)

Cluster-1 Cluster-2

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Model Mechanism: E1-HPr

X-ray crystal NMR/SAXS

Schwieters et al., JACS (2010) 132:13026-13045 (phosphorylated intermediate) Free E1 Relaxation to I through intermediate Relaxation to V through intermediate

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CNS

(direct refinement)

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Direct refinement (RDCs & SAXS) Power series expansion of SAXS curve (FAST) Rg region ---> inter-domain distance Higher angles ---> domain positions Grid-search to account for solvation

CNS: xafs.f module

Gabel et al., JBNMR (2008) 41:199-208

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CNS example

Gabel et al., JBNMR (2008) 41:199-208

SAXS + RDCs reduces possible orientations reduces inter-domain translations

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DADIMODO

(search algorithm)

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Genetic algorithm based Optimise multi-domain structures Adjusts regions of non-defined structure Start from crystal/nmr structure or homology model Objective function (SAXS & RDCs)

DADIMODO

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DADIMODO

Mareuil et al., Eur biophys J (2007) 37:95-104

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DADIMODO example

gamma-S crystallin

vertebrate eye lens component

Target Domain Models Conformers (25)

Mareuil et al., Eur biophys J (2007) 37:95-104

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DADIMODO example

gamma-S crystallin

Mareuil et al., Eur biophys J (2007) 37:95-104

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Method of intensity calculation spherical harmonics FAST, accurate (to ~ 5 nm-1) Debye SLOW, accurate (to 5-10 nm-1) Treatment of solvent Envelope Explicit layer of water Solvent treatment paramount for WAXS

Some considerations

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NMR and SAXS/SANS very complementary Scoring Direct refinement Rigid body refinement and docking But if data is rubbish --> model is rubbish! check overall parameters!!!

Summary